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European Journal of Medicinal Chemistry 43 (2008) 1774e1780 http://www.elsevier.com/locate/ejmech
Short communication
Naphthoquinoidal [1,2,3]-triazole, a new structural moiety active against Trypanosoma cruzi Eufraˆnio N. da Silva Jr. a, Rubem F.S. Menna-Barreto b, Maria do Carmo F.R. Pinto c, Raphael S.F. Silva c, Daniel V. Teixeira c, Maria Cecı´lia B.V. de Souza a, Carlos Alberto De Simone d, Solange L. De Castro b,*, Vitor F. Ferreira a, Antoˆnio V. Pinto c a
b
Departamento de Quı´mica Orgaˆnica, Instituto de Quı´mica, UFF, Campus do Valouguinho, 24020-150, Nitero´i, RJ, Brazil Laborato´rio de Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, RJ, Brazil c Nu´cleo de Pesquisas em Produtos Naturais, UFRJ, 21944-971, Rio de Janeiro, RJ, Brazil d Instituto de Quı´mica e Biotecnologia, UFAl, Tabuleiro do Martins, 57072-970 Maceio´, Alagoas, Brazil Received 25 June 2007; received in revised form 1 October 2007; accepted 11 October 2007 Available online 22 October 2007
Abstract [1,2,3]-Triazole derivatives of nor-b-lapachone were synthesized and assayed against the infective bloodstream trypomastigote form of Trypanosoma cruzi, the etiological agent of Chagas disease. All the derivatives were more active than the original quinones, with IC50/1 day values in the range of 17 to 359 mM, the apolar phenyl substituted triazole 6 being the most active compound. These triazole derivatives of nor-b-lapachone emerge as interesting new lead compounds in drug development for Chagas disease. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Chagas disease; Trypanosoma cruzi; Chemotherapy; [1,2,3]-Triazoles; Naphthoquinones
1. Introduction Chagas’ disease, caused by the parasite Trypanosoma cruzi, is endemic in Latin America [1]. The infection is transmitted by triatomine insects while blood feeding on a human host. The trypomastigote form ingested by the vector via the blood of an infected individual differentiates into epimastigote form, which, after proliferation, reaches the posterior intestine and differentiates into metacyclic trypomastigote. This latter infective form, following invasion of vertebrate cells, undergoes differentiation into amastigote, which, after several reproductive cycles, transforms into trypomastigote, responsible for the dissemination of the infection. Acute infections are usually asymptomatic, but the ensuing chronic T. cruzi infections have been associated with high ratios of morbidity and mortality [2]. Currently, treatment is unsatisfactory, being limited to
* Corresponding author. Tel.: þ55 21 2598 4330; fax: þ55 21 2598 4577. E-mail address:
[email protected] (S.L. De Castro). 0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2007.10.015
two drugs nitroheterocycles, benznidazole and nifurtimox. Their use to treat the acute phase of the disease is widely accepted, while the treatment of the chronic phase is controversial [3]. The undesirable side effects of both drugs have a major drawback in their uses, frequently forcing the abandonment of the treatment [4]. In this context, an intensive research program has been focused upon the search for alternative natural, semi-synthetic and synthetic drugs. Among several naturally occurring quinones, emerge the naphthoquinones with a broad distribution in the plant kingdom and involved in oxidative processes such as photosynthesis and electron transfer reactions [5]. In folk medicine, plants containing naphthoquinones have been employed for the treatment of many diseases, especially among Indian populations [6]. The involvement of quinones in numerous biochemical processes has led to the study of a wide variety of their synthetic derivatives. The facile reductioneoxidation of the quinone moiety appears to be the basis for the participation of a number of different quinones in electron transport and oxidative-phosphorylation processes.
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The biological activities of the naphthoquinone lapachol extracted from the heartwood of trees of the genus Tabebuia (Bigoniaceae) and its cyclization product, b-lapachone, have been intensively studied [7]. Previous work of our group showed the activity of new heterocyclic naphthoimidazole derivatives obtained from the reaction of naphthoquinones with amino-compounds [7e13]. Recently, our group also reported the synthesis and trypanocidal activity of new naphthofuranquinones [14] and of heterocyclic oxyranes [15,16]. Continuing our studies on the chemical reactivity of quinones from the Brazilian flora, we now focus on the search of compounds with naphthofuranquinoidal endowed linked to a triazolic moiety. We synthesized and characterized five derivatives of nor-lapachol (1), and assayed their activity against infective bloodstream forms of T. cruzi. The triazolic nucleus and the naphthoquinone ring are moieties with independent biological activities [5,17e19] and in this context the Huisgen cyclization under copper catalysis was employed in the present work for the obtention of naphthoquinones coupled to 1,4-triazolic nuclei and analysis and their potential trypanocidal activity was evaluated. 2. Chemistry Lapachol was extracted in large scale from the heartwood of Tabebuia sp. and purified by a series of recrystalizations, as previously described [20]. Nor-lapachol (1, 2-hydroxy-3-(20 methyl-1-propenyl)-1,4-naphthoquinone) was obtained from lapachol by Hooker oxidation, and bromo-b-nor-lapachone (2, 3-bromo-2,2-dimethyl-2,3-dihydro-naphtho[1,2-b]furan-4, 5-dione) was prepared through cyclization of 1 with bromine in chloroform. From 2, through nucleophilic substitution with sodium azide in dichloromethane was obtained the azide (3, 3azido-2,2-dimethyl-2,3-dihydro-naphtho[1,2-b]furan-4,5-dione), the key intermediate for the synthesis of quinones coupled to the triazolic nucleus, employing 1,3-dipolar reaction between the azidoquinone and an alkyne, catalyzed by Cu(I), known as ‘‘click chemistry’’. Through this type of reaction, the naphthoquinoidal triazoles 4e8 were obtained and their physical and spectroscopic data are in agreement with the structures depicted in Scheme 1. The atoms of carbons present in molecules 3 and 4e8 were differentiated using the spectra of 13 C-APT (attached proton test), where the carbons CH and CH3 are positive phases and C (quaternary) and CH2 are negative phases. The regioselectivity of the reactions was fully ascertained through X-ray crystallography study of derivative 4. 3. Results and discussion [1,2,3]-Triazoles have gained increasing attention in drug discovery since the introduction of the concept of ‘‘click’’ chemistry by Sharpless [21,22]. This kind of substances can actively participate in hydrogen bonding and dipoleedipole interactions due to their strong dipole moments, being extremely stable to hydrolysis and oxidative/reductive conditions. Several different procedures have been described for their synthesis, but the most suitable method is the 1,3-dipolar
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cycloaddition reaction [23] between substituted acetylenes and an alkyl azide derivative. This reaction originates two regioisomers, 1,4- and 1,5-triazoles. However, recently it was demonstrated that copper(I) salt’s regioselectivity promotes formation of the 1,4-triazole adduct. The improvement for this reaction was reported independently by the groups of Sharpless and Meldal [24] demonstrating that it can be carried out at room temperature in aqueous media under copper(I) catalysis. [1,2,3]-Triazoles are an important class of heterocyclic compounds due to their wide range of activities such as antiplatelet agents [25], dopamine D2 receptor ligands related to schizophrenia [26], anticonvulsants [27], anti-inflammatory, anti-allergic [28], antiviral [29] and antimicrobial agents [30]. Structure of 4 was confirmed by X-ray diffraction (Fig. 1). All the interatomic distances and angles are within the expected values for similar chemical bondings [31]. The naphthoquinonic ring is planar with maximum devia˚ ]. The distances of atoms O1 and tion for C6 [0.022(2) A C12 of furan ring to the average reference plane are ˚ and 0.006(2) A ˚ , respectively. Atoms O2 and O3 0.012(3) A ˚ are practically inside this plane with distances of 0.002(4) A ˚ and 0.012(2) A, respectively. The furan ring adopts a pure envelope conformation with Puckering parameters [q2 ¼ ˚ and 4 ¼ 292(1) ]. Atoms C12, C15, O4 and atoms 0.006(1) A of the triazole ring are all inside of the same plane of least ˚ ]. The squares with maximum distances for O4 [0.1215(4) A dihedral angle between this least squares plane and that of the naphthoquinonic ring is 89.47 . In the crystal structure packing molecules interact through strong intermolecular O4eH11/N2 hydrogen bond [symmetry code: (x 1,y,z) ˚ and O4e with O4/N2 ¼ 2.890(4), H11/N2 ¼ 1.899(3) A H11/N2 ¼ 156(2) , forming a network (Fig. 2). In order to analyze the role of the triazole substitution pattern on the furan ring moiety of 3, it was planned to prepare compounds 4e8, whose structures are dependent on the available alkynes: (a) triazoles containing polar groups (4, 5 and 7); (b) a derivative containing an aromatic apolar group, such as (6) and (c) a triazole possessing a polar group (8). The triazolic quinones 4, 5 and 7 present a hydroxyl group (leaving group), susceptible to elimination as exemplified in the Scheme 2, by elements of the biological milieu, including blood components, leading to the formation of an ammonium salt, suggesting a detoxification mechanism reducing the concentration (bioavailability) of the original compound and consequently leading to lower trypanocidal activity. Compound 8 being a hemiacetal could be hydrolyzed in aqueous medium leading to the generation of an intermediate with chemical characteristics similar to 4, 5 and 7. On the other hand, the most active compound, 6, does not present such a leaving group, not suffering similar detoxification reaction and, due to its higher lipophylic character when compared with the other triazoles, could display a better penetration through the parasite’s plasma membrane. The activity of these triazoles against trypomastigote forms of T. cruzi is shown in Table 1. All the derivatives were more active than the original quinone, nor-lapachol, being the IC50/1
E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 43 (2008) 1774e1780
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O
O
O
OH
Br2 CHCl3
Br
O
O
2
1
NaN3 CH2Cl2 O
O
O
OH
O
O
O
O
O
CuSO4.5H2O Na-ascorbate CH2Cl2/H2O
N
N N
4
O
N3
3
O
O
OH
O
CuSO4.5H2O Na-ascorbate CH2Cl2/H2O
N
N
8
N O
HO OH
CuSO4.5H2O Na-ascorbate CH2Cl2/H2O
CuSO4.5H2O Na-ascorbate CH2Cl2/H2O
CuSO4.5H2O Na-ascorbate CH2Cl2/H2O
O
N
N
N
6
O
7
N O
5´
O
HO
O
O
N
N
5
O
O OH
O
N
N N O
4´ 3´
6´
6 5a
7
5
O
4
5
4
3a
8
9a 9
1
O
2
1
3
2´
N
N
9b
1´
N
3
2 11
10
Scheme 1. Synthesis of the naphthoquinoidal triazoles obtained through 1,3-dipolar reaction with the azide intermediate 3.
Fig. 1. Projection ORTEP-3 of triazole 4.
day values in the range of 17e359 mM. Among them, the triazole 6 is the most active one. Statistical analysis showed the following order of decreasing activity ( p < 0.05): 6 > 3 ¼ 7 > 4 > 8 > 5. In the same experimental condition, the IC50/1 day value for crystal violet, the standard drug, is 536.0 3.0 mM [8] and for benznidazole 103.6 0.6. Compounds 3 and 4e8 were synthesized and assayed against the infective bloodstream trypomastigote form of T. cruzi. All the derivatives were more active than the original quinone, with IC50/1 day values in the range of 17e359 mM, showing that the triazolic nucleus acted in the increase of the biological activity, the apolar substituted triazole 6 being the most active. These triazole derivatives of nor-b-lapachone emerge as interesting new lead compounds in drug development for the treatment of Chagas disease.
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Fig. 2. Packing diagram showing the H-bonding.
4. Experimental protocols Melting points were determined on a Reichert micro hot stage and are uncorrected. Analytical grade solvents were used. The solvents were previously purified as described in the literature [32]. Column chromatography was performed on silica gel (Acros Organics 0.035e0.070 mm, pore diameter ca 6 nm). Infrared spectra were recorded on a PerkineElmer FT-IR Spectrometer. 1H NMR and 13C NMR spectra were recorded at room temperature using a Varian Unity Plus 300 instrument, in the solvents indicated, with TMS as internal standard. Chemical shifts (d) are given in ppm and coupling constants (J ) in Hertz. High-resolution electron-impact mass spectra (70 eV) were obtained using a MAT8500 instrument. The main fragments were described as a relation between atomic mass units and the charge (m/z) and the relative abundance in percentage of the base peak intensity.
4.1. Synthesis of 3-azido-2,2-dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (3) A solution of 1 (228 mg, 1 mmol) in 25 ml of dichloromethane and 2 ml bromine (26 mg, 38 mmol) was stirred until an orange precipitate was formed. To this mixture, sodium azide
(130 mg, 2 mmol) was added as a solid and stirred overnight. The azide-quinone was removed by filtration and dried at room temperature to produce 3 (269 mg, 1 mmol, 100% yield) as an orange solid (mp 200e202 C). 1H NMR (300 MHz, CDCl3) d: 8.14 (1H, ddd, J ¼ 6.9, 2.1, 0.9), 7.72e7.65 (3H, m), 4.77 (1H, s), 1.67 (3H, s), 1.55 (3H, s). 13C NMR (75 MHz, CDCl3) d: 180.3 (CaO), 175.2 (CaO), 170.2 (C0), 134.5 (CH), 132.7 (CH), 131.1 (C0), 113.5 (C0), 129.5 (CH), 125.1 (CH), 126.7 (C0), 95.5 (C0), 67.3 (CH), 27.1 (CH3), 21.9 (CH3). IR (film) nmax 1655 (CaO), 1644 (CaO), 2109 (N3) cm1. EI-HRMS (70 eV, m/z) 269.08000. Calcd for C14H11O3N3: 269.08004; (%) 227 (100), 199 (18), 104 (14), 173 (8), 76 (10), 42 (6), 50 (4), 269 (2), 181 (3), 157 (5), 128 (6).
4.2. General procedure for the synthesis of 4 to 8 Compound 3 (223.4 mg, 0.83 mmol) in 12 mL CH2Cl2/ H2O 1:1 is reacted with CuSO4$5H2O (9.3 mg, 0.04 mmol) and sodium ascorbate (22 mg, 0.11 mmol) and the desired substituted alkyne (see below). The mixture was maintained under agitation at room temperature till the total formation of the product, monitored by thin layer chromatography. The organic phase was extracted with dichloromethane, dried
+ O
OH
O O
O
N
N O
N
N N
N O
Scheme 2. Mechanism of formation of cationic species in compound 4.
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Table 1 Activity of compounds 3e8 against trypomastigote forms of T. cruzi Compound
IC50/1 day (mM)
Nor-lapachol 3 4 5 6 7 8 Crystal violet Benznidazole
1281.0 167.0a 50.2 3.8 151.9 8.0 348.1 44.2 17.3 2.0 57.8 5.6 256.7 38.7 536.0 3.0a 103.6 0.6
a
Ref. [8].
with NaSO4 and concentrated under reduced pressure. The residue obtained was purified by column chromatography on silica gel using as eluent a gradient mixture of hexane/ethyl acetate with increasing polarity up to 100% ethyl acetate. The alkynes employed were the following: for 4, 2-methylbut-3-yn-2-ol; for 5, prop-2-yn-1-ol; for 6, ethynyl-benzene; for 7, 1-ethynyl-cyclohexanol and for 8, 2-prop-2-ynyloxytetrahydro-pyran. 4.2.1. Synthesis of 3-[4-(1-hydroxy-1-methyl-ethyl)[1,2,3]triazol-1-yl]-2,2-dimethyl-2,3-dihydro-naphtho[1,2-b]furan-4,5-dione (4) Yield: 90%; orange solid; mp 220e222 C. IR (KBr) nmax 2926e2981 (CH2, CH3), 1656 (CaO), 1615 (CaO), 3423 (OH) cm1. 1H NMR (300 MHz, CDCl3) d: 5.95 (1H, s), 8.17 (1H, dd, J ¼ 6.6, 2.1), 7.82e7.64 (3H, m), 1.75 (3H, s), 1.18 (3H, s), 7.4 (1H, s), 1.59 (6H, s). 13C NMR (75 MHz, DMSO-d6) d: 95.5 (C0), 66.1 (CH), 111.3 (C0), 174.2 (CaO), 180.0 (CaO), 132.1 (C0), 134.7 (CH), 133.1 (CH), 128.8 (CH), 125.1 (CH), 126.8 (C0), 170.0 (C0), 27.1 (CH3), 20.8 (CH3), 121.0 (CH), 155.9 (C0), 67.2 (C0), 30.8 (CH3), 30.7 (CH3). EI-HRMS (70 eV, m/z) 353.13750. Calcd for C19H19O4N3: 353.13756; (%) 227 (100), 353 (12), 213 (6), 199 (18), 183 (2), 171 (7), 157 (5), 128 (7), 115 (4), 105 (3), 43 (5). 4.2.2. Synthesis of 3-(4-hydroxymethyl-[1,2,3]triazol1-yl)-2,2-dimethyl-2,3-dihydro-naphtho[1,2-b]furan4,5-dione (5) Yield: 92%; yellow solid; mp 212e214 C. IR (KBr) nmax 2937e2987 (CH2, CH3), 1655 (CaO), 1614 (CaO), 3342 (OH) cm1. 1H NMR (300 MHz, CDCl3) d: 5.97 (1H, s), 8.19 (1H, ddd, J ¼ 6.4, 2.4, 0.5), 7.81e7.73 (3H, m), 1.21 (3H, s), 1.76 (3H, s), 7.5 (1H, s), 4.77 (2H, s). 13C NMR (75 MHz, CDCl3) d: 95.3 (C0), 66.1 (CH), 111.4 (C0), 174.7 (CaO), 179.9 (CaO), 132.0 (C0), 134.7 (CH), 133.1 (CH), 128.8 (CH), 125.1 (CH), 126.7 (C0), 169.8 (C0), 20.8 (CH3), 27.1 (CH3), 123.1 (CH), 148.1 (C0), 55.2 (CH2). 4.2.3. Synthesis of 2,2-dimethyl-3-(4-phenyl-[1,2,3] triazol-1-yl)-2,3-dihydro-naphtho[1,2-b]furan-4,5-dione (6) Yield: 100%; yellow solid; mp 177e179 C. IR (KBr) nmax 2926e2980 (CH2, CH3), 1612 (CaO), 1650 (CaO) cm1. 1H
NMR (300 MHz, CDCl3) d: 6.02 (1H, s), 8.21 (1H, dd, J ¼ 6.8, 2.4), 7.84e7.71 (3H, m), 1.25 (3H, s), 1.79 (3H, s), 7.30 (1H, s), 7.84e7.33 (5H, m). 13C NMR (75 MHz, DMSO-d6) d: 95.3 (C2), 66.3 (C3), 111.1 (C3a), 174.7 (CaO), 179.8 (CaO), 132.0 (C5a), 130.6 (C1’), 134.8 (CH), 133.2 (CH), 129.1 (CH), 125.2 (CH), 126.7 (C9a), 170.0 (C9b), 21.0 (C10 or C11), 27.2 (C10 or C11), 131.7 (C5), 146.4 (C4), 129.0 (CH), 128.8 (CH), 128.7 (CH), 128.1 (CH), 125.3 (CH). 4.2.4. Synthesis of 3-[4-(1-hydroxy-cyclohexyl)[1,2,3]triazol-1-yl]-2,2-dimethyl-2,3-dihydro-naphtho[1,2-b]furan-4,5-dione (7) Yield: 90%; yellow solid; mp 186e188 C. IR (KBr) nmax 2854e2981 (CH2, CH3), 1662 (CaO), 1623 (CaO), 3372 (OH) cm1. 1H NMR (300 MHz, CDCl3) d: 5.95 (1H, s), 8.17 (1H, dd. J ¼ 6.6, 2.2), 7.82e7.69 (3H, m), 1.17 (3H, s), 1.75 (3H, s), 7.40 (1H, s), 1.96e1.52 (10H, m). 13C NMR (75 MHz, DMSO-d6) d: 95.5 (C0), 66.0 (CH), 111.3 (C0), 174.7 (CaO), 179.9 (CaO), 132.0 (C0), 134.7 (CH), 133.1 (CH), 128.8 (CH), 125.1 (CH), 127.0 (C0), 169.8 (C0), 20.7 (CH3), 27.1 (CH3), 121.5 (CH), 155.5 (C0), 68.0 (C0), 37.9 (CH2), 25.3 (CH2), 21.9 (CH2). 4.2.5. Synthesis of 2,2-dimethyl-3-[4-(tetrahydro-pyran2-yloxymethyl)-[1,2,3]triazol-1-yl]-2,3-dihydro-naphtho[1,2-b]furan-4,5-dione (8) Yield: 90%; orange solid; mp 190e192 C. IR (film) nmax 2937e2985 (CH2, CH3), 1655 (CaO), 1698 (CaO) cm1. 1 H NMR (300 MHz, CDCl3) d: 5.97 (1H, s), 8.19 (1H, dd, J ¼ 6.1, 1.9), 7.83e7.73 (3H, m), 1.20 (3H, s), 1.76 (3H, s), 7.48 (1H, s), 4.84 (1H, dd, J ¼ 12.7, 5.4), 4.63 (1H, dd, J ¼ 12.7, 3.6), 4.68 (1H, t, J ¼ 3.9), 1.95e1.46 (8H, m). 13C NMR (75 MHz, DMSO-d6) d: 95.1 (C0), 66.1 (CH), 111.3 (C0), 174.7 (CaO), 179.8 (CaO), 132.0 (C0), 134.7 (CH), 133.0 (CH), 128.7 (CH), 125.0 (CH), 126.7 (C0), 169.9 (C0), 20.7 (CH3), 27.0 (CH3), 124.1 (CH), 97.2 (CH) 143.7 (C0), 30.0 (CH2), 24.9 (CH2), 61.4 (CH2), 61.3 (CH2), 19.0 (CH2). 4.3. X-Ray analysis Crystallographic data for compound 4: C19H18N3O4; ˚, M ¼ 704.74; triclinic, space group P-1; a ¼ 7.1998(3) A ˚ , c ¼ 13.5644(12) A ˚ ; a ¼ 89.742(3) , b ¼ b ¼ 9.5301(7) A ˚ 3; Z ¼ 2; Dc ¼ 89.350(5) , g ¼ 71.586(4) ; V ¼ 883.01(11) A 1 ˚ ; F(000) ¼ 370; 1.325 g cm ; l (Mo Ka) ¼ 0.71013 A T ¼ 293 K; colorless sheet, size 0.15 0.11 0.09 mm; 5185 measured reflections, refinement based on F2 to give R1 [F2 > 2s(F2)] ¼ 0.070 and w2 ¼ 0.215 for 3730 observed reflections, and 240 parameters. The Flack absolute structure parameter was determined to be 0.8(5) and the refinement of the opposite enantiomer resulted in a value of 1.32(2). Unfortunately, the weak distinguishing value of the Flack parameter (which should be 0.0 for the correct enantiomer) cannot be used to definitively assign the absolute stereochemistry. The positions of H atoms bonded to C were determined based on
E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 43 (2008) 1774e1780
stereochemical parameters and their displacement parameters calculated as 1.5Ueq (C-methyl) or 1.2Ueq (other). X-ray data collection was accomplished on an EnrafNonius KappaCCD area-detector diffractometer. The programs used in the crystallographic study were: COLLECT [33] for data collection; HKL Denzo-Scalepack system [34] for integration and scaling of the reflections; SHELXS97 [35] for solving the structure and SHELXL-97 [35] for refining by full-matrix least squares on F2. The programs SHELXL-97 [35] and ORTEP-3 [36] were used within WinGX [37] to prepare materials for publication. A complete set of data of compound 4 has been deposited at the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 651356. 4.4. Assay for in vitro trypanocidal activity Stock solutions of the compounds (1e8) were prepared in dimethylsulfoxide (DMSO), with the final concentration of the latter in the experiments never exceeding 0.1%. Preliminary experiments showed that at concentrations of up to 0.5%, DMSO has no deleterious effect on the parasites. Bloodstream trypomastigotes of the Y strain [38] were obtained at the peak of parasitaemia from infected albino mice, isolated by differential centrifugation and resuspended in Dulbecco’s modified Eagle medium (DME) to a parasite concentration of 107 cells/ ml in the presence of 10% of mouse blood. This suspension (100 ml) was added in the same volume of each compound previously prepared at twice the desired final concentrations. Cell counts were performed in Neubauer chamber and the trypanocidal activity was expressed as IC50, corresponding to the concentration that leads to lysis of 50% of the parasites. 4.5. Statistical analysis The comparison between the IC50 values for T. cruzi was performed by ANOVA followed by the StudenteNewmane Keuls and ManneWhitney tests ( p < 0.05). Acknowledgment This research was supported by grants from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), FINEP, DECIT/SCTIE/MS, Programa de Apoio a` Pesquisa Estrate´gica em Sau´de (PAPES/FIOCRUZ), Fundac¸~ao Carlos Chagas Filho de Amparo a` Pesquisa do Rio de Janeiro (FAPERJ) and CAPES. Appendix. Supplementary data The X-ray crystallographic data of compound 4 is available free of charge on application to the Director, CCDC, 12 Union Road, Cambridge CH21EZ, UK (fax: þ44-1223-336-033 or email:
[email protected] or http://www.ccdc.cam.ac.uk). Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.ejmech.2007.10.015.
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